Antibacterial and in vivo toxicological studies of Bi2O3/CuO/GO nanocomposite synthesized via cost effective methods

In this research work, Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites have been synthesized via an eco-friendly green synthesis technique, solgel route and co-precipitation method respectively for the assessment of antibacterial activity as well as in vivo toxicity. The XRD patterns confirm the formation of Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites showing monoclinic structures. Crystallite size and lattice strain are calculated by Scherrer equation, Scherrer plot and Willimson Hall plot methods. Average crystallite size measured for Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites by Scherrer equation, Scherrer plot and WH-plot methods are (5.1, 13.9, 11.5)nm, (5.4, 14.2, 11.3)nm and (5.2, 13.5, 12.0)nm respectively. Optical properties such as absorption peaks and band-gap energies are studied by UV–vis spectroscopy. The FTIR peaks at 513 cm−1, 553 cm−1 and 855 cm−1 confirms the successful synthesis of Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites. The antibacterial activity of synthesized Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites is examined against two gram-negative (Escherichia coli and pseudomonas) as well as gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) at dose 25 mg/kg and 40 mg/kg by disk diffusion technique. Zone of inhibition for Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO at dose 40 mg/kg against E. coli (gram − ve) are 12 mm, 17 mm and 18 mm respectively and against Pseudomonas (gram − ve) are 28 mm, 19 mm and 21 mm respectively. While the zone of inhibition for Bi2O3/GO and Bi2O3/CuO/GO at dose 40 mg/kg against B. cereus (gram + ve) are 8 mm and 8.5 mm respectively and against S. aureus (gram + ve) are 5 mm and 10.5 mm respectively. These amazing results reveal that Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposite as a kind of antibacterial content, have enormous potential for biomedical applications. In addition, the in vivo toxicity of synthesized Bi2O3/CuO/GO nanocomposite is investigated on Swiss Albino mice at dose of 20 mg/kg by evaluating immune response, hematology and biochemistry at the time period of 2, 7, 14 and 30 days. No severe damage is observed in mice during whole treatment. The p value calculated by statistical analysis of hematological and biochemistry tests is nonsignificant which ensures that synthesized nanocomposites are safe and non-toxic as they do not affect mice significantly. This study proves that Bi2O3/CuO/GO nanocomposites are biocompatible and can be explored further for different biomedical applications.

www.nature.com/scientificreports/ NPs less than 5.5 nm 8 , while reticuloendothelial systems can absorb NPs of 10-15 nm range by Ist-pass extract 9,10 . Liver metabolism can partially remove nanoparticles greater than 50 nm [11][12][13][14] . Nanocomposites of metal oxides have gained significant interest in current history owing to unique morphological, photocatalytical, optical, physical, thermal, electric, absorption aspects 15,16 . Bismuth oxide (Bi 2 O 3 ) nanoparticles have recently received a lot of attention as a semiconductor material within bismuth-based products due to its simple as well as distinctive properties 17 . Bi 2 O 3 nanoparticles have several crystalline phases such as monoclinic, triclinic, tetragonal, cubic and BCC as Lopez et al. have identified several crystal phases of Bi 2 O 3 18,19 . Bismuth (Bi) is a substance with significant atomic number (Z = 83) and photoelectrical absorption co-efficient larger than Pt, I and Au. High X-ray absorbance makes it ideal for use in cancer treatment and as a contrasting agent [20][21][22][23][24] .
GO is a novel material made up of several carbon atoms layers arranged in 2D lattice [25][26][27][28][29][30] . GO have two unique regions, one with sp 2 hybrid carbon domain while second with various oxygenated groups 31 . It has promising advantages in sustainable power, electrical gadgets, transistor, photovoltaic cells and detectors among others 32 . Chemical and physical characteristics of any material are strengthened when Bi 2 O 3 is added to it 33 . Owing to literature, number of experts worked on bismuth oxide and graphene oxide composite. Das 34 .
Cu is an excellent material for antibacterial applications. Copper is now employed as antibiotic, antifungicide as well as antifouling agent 35 . It was discovered that metal surfaces having copper are now the most efficient in lowering bacterial growth after examining a variety of metal surfaces 36,37 . Copper oxide is p-type semiconductor having 1.2 eV bandgap, has received significant interest in low price, processability, wide surface area and renewability 38,39 . CuO nanoparticles are good contender for making antimicrobial medical equipment, bandages and ointments 40 . CuO nanoparticles have been used in variety of biological studies. Booshehri et al. investigate that copper oxide nanoparticles have higher antibacterial property 41 . CuO nanoparticles have been found to have anticancer properties in A549 lung cancer cells 42 . From historical eras copper and copper oxides have been used in numerous biomedical applications such as tissue repair, grocery bags, and dental standards and so on, owing to unique antibacterial and anticancer abilities 43,44 . Nanocomposites are in demand in hopes of improving biological efficiency while also meeting specific requirements. As a result, GO can provide an appropriate platform for functionalizing or hosting CuO nanoparticles. CuO and GO might be a fruitful combination of two material's properties leading to a revolutionary series of nanocomposites exhibiting unique properties. As a result, we discovered these hybrid composites worth investigating in our search of materials with improved biological activity (anticancer and antibacterial property) 45,46 .
The current work is aimed on developing an easy method for producing Bi 2 O 3 /GO and Bi 2 O 3 /CuO/GO nanocomposites to improve biomedical applications. The produced nanocomposites are analyzed using number of physical techniques including XRD, UV-vis, SEM and FTIR. Our synthesized nanocomposites are theranostic as well as antibacterial agents. Synthesized nanocomposites have many biological applications and mainly utilized as antibacterial agents. They are used for diagnostic and treatment purpose as well as used in hospitals to kill bacteria as antibacterial agents. Escherichia coli and Pseudomonas bacteria are used to investigate the antibacterial activity of synthesized nanocomposites. Synthesis of Bi 2 O 3 nanoparticles. 30 g of washed mentha leaves were immersed in 300 ml of distilled water and heated for 2 h at 80 °C to prepare leaf extract for the synthesis of bismuth oxide. Leaf extract was cooled at room temperature before being filtered through Whatman filter paper. 3 g of bismuth nitrate was dissolved in 15 ml of distilled water at 80 °C and also combined with 30 ml of prepared extract at 80 °C and stirred continuously. After 20 h, Bi 2 O 3 nanoparticles were obtained. Sample was washed with distilled water as well as dried. Resulted product was heated in furnace at 500 °C for 4 h to remove the impurities.    www.nature.com/scientificreports/ Scherrer plot method. Scherrer plot methodology was used to investigate the widening of peaks along lattice strain or crystallite size related to dislocation using XRD 52 . Bragg's peak width is equal to summation of instrumental and sample effect, as calculated by the formula:

Synthesis of
By taking 1/β along x-axis as well as Cosθ along y-axis Scherrer plot was drawn as shown in Fig. 2a. After linear fitting of data, crystal size was estimated from slope of liner line.
Williamson Hall (WH) plot method. The W-H analysis is dependent on the assumption that the estimated formulas for strain widening " β s " as well as size broadening " β D " change in opposite directions when Bragg's angle (θ) is taken into account. Due to crystal defects and deformation, straininduce widening can occur which is given as.   www.nature.com/scientificreports/ Unlike Scherrer plot method, WH method is independent on 1/cosθ but dependent on tanθ. With variation in micro strain and crystal size in the crystal, the difference in 2θ allows us to distinguish b/w strain and size influences on peak widening.
By adding strain and crystallite size total peak broadening is attained.
Furthermore, the uniform deformation model is used in WH analysis to assume the micro strain to be equal in all crystallographic orientations 52 . β hkl for this model is By multiplying cosθ on both sides.
Here "ε" is strain. WH plots of are drawn by taking 4sinθ on x-axis and βcosθ on y-axis as shown in Fig. 2b. Micro strain is measured from slope while crystal size is calculated from intercept of linear fitted values 53 . Crystal size by all the three methods WH plot, Scherrer formula and Scherrer plot is shown in Table 2.
FTIR analysis. FTIR was used to study functional groups and significance of multiple kinds of functional groups within infrared spectra. FTIR analysis of NPs revealed a number of absorption peaks ranging from 4000 to 400 cm −1 as displayed in Fig. 3. FTIR graphs are plotted b/w wavelength (cm −1 ) on x-axis and transmittance (a.u) on y-axis. Figure 62,63 . In (c) Bi 2 O 3 /GO, composite peak of GO and Bi 2 O 3 is present which confirms that both oxides exist in single matrix. In graph (d), CuO peak is present at 280 nm as well as GO and bismuth oxide peak is also present which confirms that three oxides exist in one matrix 64 . Single oxide phase scattering caused these absorption peaks in nanocomposite. The extended tail in the absorption spectrum's wavelength range is most likely caused by scattered radiations from mixed oxide nanoparticles.

Bandgap determination. Optical bandgap energy is estimated by this relation:
A is characteristic factor; υ is frequency of incident light. Bandgap energy is estimated by plotting graph b/w hυ on x-axis and (αhυ) 2 on y-axis.    www.nature.com/scientificreports/ In Fig. 5a  UV analysis revealed that bandgap energy of nanocomposite may be controlled by adjusting volume fraction of the material for a variety of applications including solar cells, solid oxide and photocatalytical activity. SEM analysis. The GO structure is sheet like and functional groups which contain oxygen may interact with GO layers and cause folding of GO sheets 65 . In Fig. 6a, GO sheet like structure was observed in nanocomposite containing well-dispersed Bi 2 O 3 on the surface 66 . The image shows that Bi 2 O 3 /GO nanocomposite surface is rough, may be due to development of NPs of Bi 2 O 3 on GO sheets 67 . In Fig. 6b, CuO NPs were distributed ran-   EDX analysis. Figure 7 shows the EDX of Bi 2 O 3 /GO in which carbon which represents the GO is present.
Bismuth is also present in the EDX of Bi 2 O 3 /GO. Similarly, in the EDX of Bi 2 O 3 /CuO/GO bismuth and copper are present with GO which is shown in Fig. 8.  Fig. 9a and b. Inhibitory zone of pseudomonas and E. coli (gram − ve) bacteria measured in mm is comparably higher than B. cereus and S. aureus (gram + ve) bacteria. Inhibition zone of Bi 2 O 3 /CuO/GO composite is higher than Bi 2 O 3 /GO nanocomposite as shown in Fig. 9a and b. Moreover, the scattering of metal oxides via GO sheets improves the antibacterial property 69 . Figure 10 shows the antibacterial activity of Bi 2 O 3 against E. coli and pseudomonas bacteria. According to Table 3 Table 4. Almost all the antibiotic discs of Fox 30 shows 18 mm zone of inhibition shown in Figs. 13a-d and 14a and c, while Fig. 14b and c shows zone of inhibition greater than 18 mm. Figures 11 and 12 demonstrate that our sample shows better results than Erythromycin against B. cereus and S. aureus bacteria.

In vivo toxicity studies
The in vivo toxicity of Bi 2 O 3 /CuO/GO at 20 mg/kg dose was explored on Swiss Albino mice (female) by analyzing body weight, acute toxicity study, hematological as well as biochemistry test at 2, 7, 14 and 30 days. Animals were divided into two groups. One is control group and second is treated group. Each group contain 4 animals. 20 mg/kg dose was administered orally to all the mice of treated group. One mouse from each group was slaughtered at 2, 7, 14 and 30 days. Blood samples and organs (liver, lungs and kidneys) were collected to perform hematological, biochemistry and pathological test. The treatment with Bi 2 O 3 /CuO/GO had no clear deleterious effects on the growth during 30 days period, no immortality was found and there were no significant differences in weight of body b/w Bi 2 O 3 /CuO/GO treated mice and control mice. The acute toxicity parameters such as Alertness, convulsions, grooming, hyperactivity, salivation, lacrimation, sweating, urination, righting reflex, gripping strength, corneal reflex, writhing reflex and pain response were investigated at 30-day time period and no meaningful difference was noticed. The animal studies were performed according to the ARRIVE guidelines. Additionally, all experimental protocols and animal care procedures were according to the guidelines approved  www.nature.com/scientificreports/ www.nature.com/scientificreports/    www.nature.com/scientificreports/ as they are strongly linked to liver as well as kidney function of mice. After 2 days of treatment no significant changes were noticed in ALT and CREA but AST was decreased. After 2 days, NPs caused significant liver inflation but did not trigger open wound. No significant change was noticed in ALT and AST after 7 days and 14 days of treatment but CREA was increased after 14 days. ALT and AST have no significant change after 30 days as well as CREA turned to normal level, indicating that minor damage of liver caused by Bi 2 O 3 /CuO/GO was repaired after 30 days. This is accordance with removal and biodistribution of NPs. The accumulation of oxide-based and Au NPs in liver have been demonstrated to cause a steady increase in ALT and AST as well as substantial liver damage. Au NPs with 5 mg/kg dose level via intravenous administration cause severe injury 79,80 . BUN was slightly increased after 30 days. ALB, cholesterol, sugar, TBIL, total protein, triglycerides and Uric acid were also investigated but no significant change was noticed after 30 days. Pathological tests results. We use immunohistochemistry to examine the pathogenic alterations in organs like liver, lungs and kidneys at time period of 2, 7, 14 and 30 days. We collected these organs and prepared the slides of tissues of these organs for microscopy. Throughout the whole treatment session, no damage was identified in kidneys shown in Fig. 17. There was a minor pathological change in liver and lungs was noticed. Very small dark spot was noticed in liver after 4 days but it returned to normal after 30 days. Similarly, a small dark spot was identified in lungs after 2 and 14 days but recovered in 30 days. Additional element of pathology is to analyze the removal of Bi 2 O 3 /CuO/GO NPs qualitatively 81,83 . Whenever NPs assemble in organs, they can accumulate and be recovered directly by using optical microscope. Mice treated with Fe 3 O 4 NPs, GO and carbon nanotubes had comparable effects. www.nature.com/scientificreports/ The liver was found to have black spots, which vanished after roughly 90 days. Now days, Nanomedicine is looking for compounds which have low toxicity and show high clearance efficiency. NPs of small size are commonly believed to be removed through kidneys. But reality on the other hand is significantly more complicated than the assumptions. The clearing of Au NPs is a good example. Au NPs of size 3 nm protected with PEG were not cleared, but Au NPs of same size protected with glutathione were cleared efficiently 79,84 . When the size of carbon materials was between 10 and 30 nm, the kidney and liver may progressively eliminate them 83 . As a result, it is obvious that NPs clearance is influenced not just by size and morphology, but also by shape and durability. Another feasible path for the construction of metabolizable NPs is to investigate the removal of materials of large size. Our current study conclusively demonstrates that Bi 2 O 3 /CuO/GO nanocomposites may be absorbed by liver, indicating that they hold a great potential in medical uses including cancer treatment and contrast agents 82 .
Kefayat et al. 85 reported the toxicity of albumin stabilized GNPs on BALB/c mice which were injected intra venously with 10 mg/kg dose and sacrificed after 1 month. Through histopathological and biochemistry blood and biochemistry blood analysis as well as histopathological images of organs (liver, lungs, brain, spleen, heart and kidneys) it was observed that GNPs were safe and non-toxic 85 . Toxicity studies of FA-AUNCs on rats with 10 mg/kg dosage injected via intra venous route for a time period of 3 days were reported by Kefayat et al. 86 . By biochemistry analysis and histopathological assays of organs (kidneys, liver and spleen), they concluded that no severe damage was found 86 . Ghahremani et al. 87 , reported that APT-GNCs exhibit no toxic effect on BALB/c mice in a duration of 20 days when a dose of 8 mg/kg was injected intravenously to them. Tables 5 and 6 shows the p value of hematological and biochemistry parameters respectively. GraphPad (2D scientific Graphing) software (version 8) was used to calculate p value. From p value it is very clear that synthesized nanoparticles have no significant effect on mice and our nanoparticles are safe. p Value shows non-significant results. Similarly, Table 7 shows acute toxicity study of mice at 0 h, 2 h, 6 h, 24 h and 48 h and there is no significant change occurs in behavior of mice.   www.nature.com/scientificreports/  Pathological results show that toxicity was induced in both liver and kidneys. In liver, necrosis of tissues and in kidney necrosis in proximal renal tubule as well a swelling of proximal tubule was observed 89 In vivo CuO NPs Male wister rats 10, 100 and 300 mg/kg dose delivered through IP injection for 14 days

Statistical analysis.
Toxicity was induced in lungs and liver with all concentration of CuO NPs. In liver, vasculature in central veins, portal triad vessels and loss of hexagonal lobules was observed. And in lungs thickening of air scars can be seen 90